Canadian Journal of Physiology and Pharmacology, 2015
Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it ... more Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p < 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.
Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction ... more Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by matrix metalloproteinase (MMP)-9. Here, we report a cardiac aging signature that encompasses macrophage pro-inflammatory signaling in the left ventricle (LV) and distinguishes biological from chronological aging. Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac aging signature. The decline in early to late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, while LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages a...
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial i... more Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial infarction (MI). MMP-12 has potent macrophage-dependent remodeling properties in the atherosclerotic plaque; however, post-MI roles have not been examined. The goal was to determine MMP-12 post-MI mechanisms. Male C57BL/6J mice (3-6months old) were subjected to left coronary artery ligation. Saline or the RXP 470.1 MMP-12 inhibitor (MMP-12i; 0.5mg/kg/day) was delivered by osmotic mini-pump beginning 3h post-MI, and mice were sacrificed at day (d)1, 3, 5 or 7 post-MI and compared to d0 controls (mice without MI; n=6-12/group/time). MMP-12 expression increased early post-MI, and contrary to expected, neutrophils were a surprising early cellular source for MMP-12. MMP-12i reduced MMP-12 activity 33±1% at d1 post-MI. Despite similar infarct areas and survival rates, MMP-12i led to greater LV dilation and worsened LV function. At d7 post-MI, MMP-12i prolonged pro-inflammatory cytokine upregulation (IL1r1, IL6ra, IL11, and Cxcr5) and decreased CD44 (both gene and protein levels). Hyaluronan (HA), a CD44 ligand, was elevated at d1 and d7 post-MI with MMP12i, as a result of decreased fragmentation. Because CD44-HA regulates neutrophil removal, apoptosis markers were evaluated. Caspase 3 increased, while cleaved caspase 3 levels decreased in MMP-12i group at d7 post-MI, indicating reduced neutrophil apoptosis. In isolated neutrophils, active MMP-12 directly stimulated CD44, caspase 3, and caspase 8 expression. Our results reveal a novel protective mechanism for MMP-12 in neutrophil biology. Post-MI, MMP-12i impaired CD44-HA interactions to suppress neutrophil apoptosis and prolong inflammation, which worsened LV function.
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
Journal of molecular and cellular cardiology, Jan 12, 2015
Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the ... more Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the left ventricle (LV) post-myocardial infarction (MI); however the biomechanical mechanisms underlying the improved outcome are poorly understood. The aim of this study was to determine the mechanisms whereby MMP-9 deletion alters collagen network composition and assembly in the LV post-MI to modulate the mechanical properties of myocardial scar tissue. Adult C57BL/6J wild-type (WT; n=88) and MMP-9 null (MMP-9(-/-); n=92) mice of both sexes underwent permanent coronary artery ligation and were compared to day 0 controls (n=42). At day 7 post-MI, WT LVs displayed a 3-fold increase in end-diastolic volume, while MMP-9(-/-) showed only a 2-fold increase (p<0.05). Biaxial mechanical testing revealed that MMP-9(-/-) infarcts were stiffer than WT infarcts, as indicated by a 1.3-fold reduction in predicted in vivo circumferential stretch (p<0.05). Paradoxically, MMP-9(-/-) infarcts had a 1....
Canadian Journal of Physiology and Pharmacology, 2015
Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it ... more Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.
... Author; LINDSEY ML (Medical Univ. South Carolina, Sc) ESCOBAR GP (Medical Univ. South Carolin... more ... Author; LINDSEY ML (Medical Univ. South Carolina, Sc) ESCOBAR GP (Medical Univ. South Carolina, Sc) CHAPMAN RE (Medical Univ. South Carolina, Sc) PATRICK DK (Medical Univ. South Carolina, Sc) HENDRICK JW (Medical Univ. ...
Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction ... more Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by matrix metalloproteinase (MMP)-9. Here, we report a cardiac aging signature that encompasses macrophage pro-inflammatory signaling in the left ventricle (LV) and distinguishes biological from chronological aging. Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac aging signature. The decline in early to late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, while LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages a...
American journal of physiology. Cell physiology, Jan 15, 2015
To investigate the role of SPARC in age-related cardiac inflammation, we studied 6 groups of mice... more To investigate the role of SPARC in age-related cardiac inflammation, we studied 6 groups of mice: young (3-5 month old), middle-aged (10-12 month old) and old (18-29 month old) C57BL/6 wild type (WT) and SPARC-null (Null) mice (n=7-10/group). Cardiac function and structure were determined by echocardiography. The left ventricle (LV) was used for cytokine gene array and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n=5-6, p<0.05 for young vs. old), but not in Null. Pro-inflammatory markers (Ccl5, Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT, whereas they were increased only in old Null compared to respective young (n=5-6, p<0.05 for all). These results suggest that SPARC deletion delayed age-related cardiac inflammation. To further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n=4). SPARC treatment increased expression of pro-inflammatory macrophage M1 markers and d...
Canadian Journal of Physiology and Pharmacology, 2015
Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it ... more Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.
Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction ... more Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by matrix metalloproteinase (MMP)-9. Here, we report a cardiac aging signature that encompasses macrophage pro-inflammatory signaling in the left ventricle (LV) and distinguishes biological from chronological aging. Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac aging signature. The decline in early to late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, while LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages a...
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial i... more Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial infarction (MI). MMP-12 has potent macrophage-dependent remodeling properties in the atherosclerotic plaque; however, post-MI roles have not been examined. The goal was to determine MMP-12 post-MI mechanisms. Male C57BL/6J mice (3-6months old) were subjected to left coronary artery ligation. Saline or the RXP 470.1 MMP-12 inhibitor (MMP-12i; 0.5mg/kg/day) was delivered by osmotic mini-pump beginning 3h post-MI, and mice were sacrificed at day (d)1, 3, 5 or 7 post-MI and compared to d0 controls (mice without MI; n=6-12/group/time). MMP-12 expression increased early post-MI, and contrary to expected, neutrophils were a surprising early cellular source for MMP-12. MMP-12i reduced MMP-12 activity 33±1% at d1 post-MI. Despite similar infarct areas and survival rates, MMP-12i led to greater LV dilation and worsened LV function. At d7 post-MI, MMP-12i prolonged pro-inflammatory cytokine upregulation (IL1r1, IL6ra, IL11, and Cxcr5) and decreased CD44 (both gene and protein levels). Hyaluronan (HA), a CD44 ligand, was elevated at d1 and d7 post-MI with MMP12i, as a result of decreased fragmentation. Because CD44-HA regulates neutrophil removal, apoptosis markers were evaluated. Caspase 3 increased, while cleaved caspase 3 levels decreased in MMP-12i group at d7 post-MI, indicating reduced neutrophil apoptosis. In isolated neutrophils, active MMP-12 directly stimulated CD44, caspase 3, and caspase 8 expression. Our results reveal a novel protective mechanism for MMP-12 in neutrophil biology. Post-MI, MMP-12i impaired CD44-HA interactions to suppress neutrophil apoptosis and prolong inflammation, which worsened LV function.
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
Journal of molecular and cellular cardiology, Jan 12, 2015
Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the ... more Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the left ventricle (LV) post-myocardial infarction (MI); however the biomechanical mechanisms underlying the improved outcome are poorly understood. The aim of this study was to determine the mechanisms whereby MMP-9 deletion alters collagen network composition and assembly in the LV post-MI to modulate the mechanical properties of myocardial scar tissue. Adult C57BL/6J wild-type (WT; n=88) and MMP-9 null (MMP-9(-/-); n=92) mice of both sexes underwent permanent coronary artery ligation and were compared to day 0 controls (n=42). At day 7 post-MI, WT LVs displayed a 3-fold increase in end-diastolic volume, while MMP-9(-/-) showed only a 2-fold increase (p<0.05). Biaxial mechanical testing revealed that MMP-9(-/-) infarcts were stiffer than WT infarcts, as indicated by a 1.3-fold reduction in predicted in vivo circumferential stretch (p<0.05). Paradoxically, MMP-9(-/-) infarcts had a 1....
Canadian Journal of Physiology and Pharmacology, 2015
Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it ... more Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.
... Author; LINDSEY ML (Medical Univ. South Carolina, Sc) ESCOBAR GP (Medical Univ. South Carolin... more ... Author; LINDSEY ML (Medical Univ. South Carolina, Sc) ESCOBAR GP (Medical Univ. South Carolina, Sc) CHAPMAN RE (Medical Univ. South Carolina, Sc) PATRICK DK (Medical Univ. South Carolina, Sc) HENDRICK JW (Medical Univ. ...
Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction ... more Cardiac aging involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by matrix metalloproteinase (MMP)-9. Here, we report a cardiac aging signature that encompasses macrophage pro-inflammatory signaling in the left ventricle (LV) and distinguishes biological from chronological aging. Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac aging signature. The decline in early to late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, while LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages a...
American journal of physiology. Cell physiology, Jan 15, 2015
To investigate the role of SPARC in age-related cardiac inflammation, we studied 6 groups of mice... more To investigate the role of SPARC in age-related cardiac inflammation, we studied 6 groups of mice: young (3-5 month old), middle-aged (10-12 month old) and old (18-29 month old) C57BL/6 wild type (WT) and SPARC-null (Null) mice (n=7-10/group). Cardiac function and structure were determined by echocardiography. The left ventricle (LV) was used for cytokine gene array and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n=5-6, p<0.05 for young vs. old), but not in Null. Pro-inflammatory markers (Ccl5, Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT, whereas they were increased only in old Null compared to respective young (n=5-6, p<0.05 for all). These results suggest that SPARC deletion delayed age-related cardiac inflammation. To further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n=4). SPARC treatment increased expression of pro-inflammatory macrophage M1 markers and d...
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